Single chain antibody and use thereof

ABSTRACT

The present invention provides a single chain antibody that retains its original specific binding activity with an antigen, and a labeled single chain antibody in which a labeling substance is bound to the single chain antibody. Specifically, the labeled single chain antibody of the present invention can be produced by linking a labeling substance to a linker part of a single chain antibody. The antibody is produced using a wheat embryo-derived cell-free protein synthesis system, and production is carried out in a low reductive state that allows an intramolecular disulfide bond to be retained. Further, bonding the antibody to a solid phase via the labeling substance enables production of an immobilized single chain antibody as well as a method for analyzing an antigen-antibody reaction using the immobilized single chain antibody.

This application claims the benefit of priority from Japanese PatentApplication No. 2002-210067, the entire contents of which areincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a single chain antibody having astructure in which a heavy chain and a light chain of the antibody arecrosslinked through a linker, a labeled single chain antibody in which alabeling substance is provided in a linker part of the aforementionedantibody, and methods for utilizing the same.

2. Description of the Related Art

A single chain antibody is small in size in comparison to a complete IgGsince it comprises only an antigen-binding region, and thus a featurethereof is that non-specific binding to a cell can be lessened. Whenusing a single chain antibody for analysis of an antigen-antibodyreaction, a method has been developed in which various labels areattached to antibodies for the purpose of tracking immunoreaction(Cloutier, S. M. et al., Mol. Immunol., 37, 1067-1077 (2000)). Althoughvarious methods have been proposed for labeling an antibody, such as amethod in which biotin or the like is bound to the C terminus or Nterminus of the antibody using a biotin ligase (Cloutier, S. M. et al.,Mol. Immunol., 37, 1067-1077 (2000)), a problem has existed in that theactivity of the antibody to bind with an antigen is reduced by thelabel.

In recent years, the development of techniques for immobilizing thiskind of antibody on chips or beads or the like for the purpose ofdetecting specific antigens present on a cell surface rapidly and inlarge amounts has also been remarkable (Mitchell, P., NatureBiotechnology, 20, 225-229 (2002)). More specifically, while techniquessuch as microspotting, microprinting, and chemical modification areused, each of these has problems that the binding activity of theantibody to an antigen is lowered, the high-density application isdifficult, and the like.

Meanwhile, a method has also been proposed in which substances havingspecific binding ability such as streptavidin/biotin that covalentlybind to immobilized protein reaction plates are bonded as linkers.However, in these methods also, no examples exist in which animmobilized antibody maintained its binding ability against the antigen.

SUMMARY OF THE INVENTION

It is an object of this invention to provide an antibody in which asingle chain antibody having a structure in which a heavy chain and alight chain of the antibody are crosslinked through a linker maintainsbinding activity with an antigen, a labeled single chain antibodyproduced by labeling the aforementioned antibody, and methods thatutilize these. A further object of this invention is to provide a methodfor immobilizing an antibody while maintaining the binding ability ofthe antibody against its antigen, a labeled single chain antibody foruse in the method, and a method for analyzing an antigen-antibodyreaction that uses the labeled single chain antibody.

After conducting concentrated research to solve the above-describedproblems, the present inventors bound biotin to the linker part of asingle chain antibody in which the heavy chain and the light chain ofthe antibody were connected through a linker, and brought the singlechain antibody into contact with a reaction plate whose surface wascoated with streptavidin to bind the antibody to the reaction plate.When we brought an antigen into contact with the immobilized singlechain antibody produced in this manner, we found that the bindingability of the antibody against the antigen was maintained at anextremely high level. This invention was accomplished based on thesefindings.

More specifically, the present invention provides the following:

-   1. A single chain antibody comprising having a structure in which a    heavy chain and a light chain of the antibody are crosslinked    through a linker, or a labeled single chain antibody comprising    carrying a labeling substance in the linker part of the single chain    antibody.-   2. A single chain antibody having a structure in which a heavy chain    and a light chain of the antibody are crosslinked through a linker,    or a labeled single chain antibody carrying a labeling substance in    the linker part of the single chain antibody, wherein the heavy    chain and the light chain of the antibody are variable regions.-   3. A labeled single chain antibody having a structure in which a    heavy chain and a light chain of the antibody are crosslinked    through a linker, and carrying a labeling substance in the linker    part, wherein the labeling substance is a substance that is capable    of binding to a polypeptide of the linker part of the antibody in    the presence of a specific enzyme.-   4. A labeled single chain antibody having a structure in which a    heavy chain and a light chain that are variable regions of the    antibody are crosslinked through a linker, and carrying a labeling    substance in the linker part, herein the labeling substance is a    substance that is capable of binding to a polypeptide of the linker    part of the antibody in the presence of a specific enzyme.-   5. A labeled single chain antibody having a structure in which a    heavy chain and a light chain of the antibody are crosslinked    through a linker, and carrying a labeling substance in the linker    part, wherein the labeling substance is incorporated as one part of    the linker part of the antibody.-   6. A labeled single chain antibody having a structure in which a    heavy chain and a light chain that are variable regions of the    antibody are crosslinked through a linker, and carrying a labeling    substance in the linker part, wherein the labeling substance is    incorporated as one part of the linker part of the antibody.-   7. A labeled single chain antibody having a structure in which a    heavy chain and a light chain of the antibody are crosslinked    through a linker, and carrying in the linker part a labeling    substance that is capable of binding to a polypeptide of the linker    part of the antibody in the presence of a specific enzyme, wherein    the labeling substance is biotin and the enzyme is a biotin ligase.-   8. A labeled single chain antibody having a structure in which a    heavy chain and a light chain that are variable regions of the    antibody are crosslinked through a linker, and carrying in the    linker part a labeling substance that is capable of binding to a    polypeptide of the linker part of the antibody in the presence of a    specific enzyme, wherein the labeling substance is biotin and the    enzyme is a biotin ligase.-   9. The single chain antibody or labeled single chain antibody    according to any one of the above 1 to 8, which has a Kd value that    is equivalent to a Kd value of a naturally occurring antibody and    which was produced by a cell-free protein translation system using    wheat embryo.-   10. A DNA, wherein DNAs encoding a heavy chain and a light chain of    an antibody having binding ability against a specific antigen are    linked through a DNA encoding a linker.-   11. A DNA in which DNAs encoding a heavy chain and a light chain of    an antibody having binding ability against a specific antigen are    linked through a DNA encoding a linker, wherein the heavy chain and    the light chain of the antibody are variable regions.-   12. A DNA in which DNAs encoding a heavy chain and a light chain of    an antibody having binding ability against a specific antigen are    linked through a DNA encoding a linker, wherein the DNA encoding a    linker comprises a nucleotide sequence that is capable of binding    with a labeling substance in the presence of a specific enzyme after    translation.-   13. A DNA in which DNAs encoding a heavy chain and a light chain    that are variable regions of an antibody having binding ability    against a specific antigen are linked through a DNA encoding a    linker, wherein the DNA encoding a linker comprises a nucleotide    sequence that is capable of binding with a labeling substance in the    presence of a specific enzyme after translation.-   14. A DNA in which DNAs encoding a heavy chain and a light chain of    an antibody having binding ability against a specific antigen are    linked through a DNA encoding a linker comprising a nucleotide    sequence that is capable of binding with a labeling substance in the    presence of a specific enzyme after translation, wherein the    nucleotide sequence that is capable of binding with a labeling    substance encodes an amino acid sequence that is recognized by a    biotin ligase.-   15. A DNA in which DNAs encoding a heavy chain and a light chain    that are variable regions of an antibody having binding ability    against a specific antigen are linked through a DNA encoding a    linker comprising a nucleotide sequence that is capable of binding    with a labeling substance in the presence of a specific enzyme after    translation, wherein the nucleotide sequence that is capable of    binding with a labeling substance encodes an amino acid sequence    that is recognized by a biotin ligase.-   16. A method for producing a labeled single chain antibody, wherein    the DNA of any of the preceding 10 to 15 is subject to transcription    and translation using a protein synthesis system in the presence of    a labeling substance and a specific enzyme.-   17. A method for producing a single chain antibody or a labeled    single chain antibody, wherein the DNA of either of the foregoing 10    or 11 is subject to transcription and translation using a protein    synthesis system.-   18. The method for producing a single chain antibody or a labeled    single chain antibody according to the preceding 16 or 17, wherein    the protein synthesis system is a wheat embryo-derived cell-free    protein translation system, and a concentration of a reducing agent    in a translation reaction solution thereof is a concentration at    which a disulfide bond of a single chain antibody to be produced is    maintained and cell-free protein synthesis is enabled.-   19. The method for producing a single chain antibody or a labeled    single chain antibody according to the preceding 18, wherein the    method is conducted in the presence of an enzyme that catalyzes a    disulfide bond exchange reaction.-   20. A single chain antibody or a labeled single chain antibody    having a Kd value that is equivalent to a Kd value of a naturally    occurring antibody, wherein the single chain antibody or the labeled    single chain antibody is produced by the method for producing a    single chain antibody or labeled single chain antibody according to    the preceding 19 using a wheat embryo-derived cell-free protein    translation system.-   21. A method for producing an immobilized single chain antibody,    wherein any one of the antibodies described hereunder is contacted    with a reaction plate compartmentalized into a plurality of regions    having on the surface thereof a substance that binds specifically    with a labeling substance of the antibody:-   (1) a labeled single chain antibody, wherein the antibody has a    structure in which a heavy chain and a light chain of the antibody    are crosslinked through a linker and the antibody carries a labeling    substance in the linker part;-   (2) a labeled single chain antibody having a structure in which a    heavy chain and a light chain of the antibody are crosslinked    through a linker, and carrying a labeling substance in the linker    part, wherein the heavy chain and the light chain of the antibody    are variable regions;-   (3) a labeled single chain antibody having a structure in which a    heavy chain and a light chain of the antibody are crosslinked    through a linker, and carrying a labeling substance in the linker    part, wherein the labeling substance is a substance that is capable    of binding to a polypeptide of the linker part of the antibody in    the presence of a specific enzyme;-   (4) a labeled single chain antibody having a structure in which a    heavy chain and a light chain that are variable regions of the    antibody are crosslinked through a linker, and carrying a labeling    substance in a linker part, wherein the labeling substance is a    substance that is capable of binding to a polypeptide of the linker    part of the antibody in the presence of a specific enzyme;-   (5) a labeled single chain antibody having a structure in which a    heavy chain and a light chain of the antibody are crosslinked    through a linker, and carrying a labeling substance in the linker    part, wherein the labeling substance is incorporated as one part of    the linker part of the antibody;-   (6) a labeled single chain antibody having a structure in which a    heavy chain and a light chain that are variable regions of the    antibody are crosslinked through a linker, and carrying a labeling    substance in the linker part, wherein the labeling substance is    incorporated as one part of the linker part of the antibody;-   (7) a labeled single chain antibody having a structure in which a    heavy chain and a light chain of the antibody are crosslinked    through a linker, and carrying in the linker part a labeling    substance that is capable of binding to a polypeptide of the linker    part of the antibody in the presence of a specific enzyme, wherein    the labeling substance is biotin and the enzyme is a biotin ligase;-   (8) a labeled single chain antibody having a structure in which a    heavy chain and a light chain that are variable regions of the    antibody are crosslinked through a linker, and carrying in a linker    part a labeling substance that is capable of binding to a    polypeptide of the linker part of the antibody in the presence of a    specific enzyme, wherein the labeling substance is biotin and the    enzyme is a biotin ligase.-   22. A method for producing an immobilized single chain antibody    according to the method described in the preceding 21, wherein two    or more kinds of different immobilized single chain antibodies are    immobilized on a reaction plate compartmentalized into a plurality    of regions.-   23. The production method according to the preceding 21 or 22,    wherein a labeling substance is biotin and a substance that binds    specifically with the labeling substance is streptavidin.-   24. An immobilized single chain antibody prepared by the production    method according to any one of the preceding 21 to 23.-   25. A method for analyzing an antigen-antibody reaction, wherein a    test substance is contacted with the immobilized single chain    antibody according to the preceding 24, and binding ability of the    test substance against the immobilized single chain antibody is    analyzed.-   26. A method for analyzing an antigen-antibody reaction, comprising    the steps of:-   (1) preparing a labeled single chain antibody under conditions in    which a disulfide bond of a single chain antibody is retained,    comprising the step of the following (i) or (ii):-   (i) producing a labeled single chain antibody by subjecting a DNA,    in which DNAs encoding a heavy chain and a light chain of an    antibody having binding ability with a specific antigen are linked    through a DNA encoding a linker comprising a nucleotide sequence    that is capable of binding with a labeling substance in the presence    of a specific enzyme after translation, to transcription and    translation using a wheat cell-free protein synthesis system in the    presence of a specific enzyme; or-   (ii) producing a labeled single chain antibody by subjecting a DNA,    in which DNAs encoding a heavy chain and a light chain that are    variable regions of an antibody having binding ability with a    specific antigen are linked through a DNA encoding a linker    comprising a nucleotide sequence that is capable of binding with a    labeling substance in the presence of a specific enzyme after    translation, to transcription and translation using a wheat    cell-free protein synthesis system in the presence of a specific    enzyme;-   (2) preparing a substance (adapter substance) that binds    specifically with a labeling substance of a labeled single chain    antibody in a case where the labeling substance of the labeled    single chain antibody is an immobilizing substance, comprising the    steps of:-   (i) immobilizing a substance (adapter substance) that binds    specifically with a labeling substance of a labeled single chain    antibody on a reaction plate compartmentalized into a plurality of    regions;-   (ii) removing the substance (adapter substance) that binds    specifically with a labeling substance of a labeled single chain    antibody that was not immobilized to the reaction plate in the    preceding (i); and-   (iii) before and after the step of the preceding (i) or (ii),    removing nonspecific adsorption from the reaction plate as    appropriate;-   (3) preparing an immobilized labeled single chain antibody in a case    where a labeling substance of the labeled single chain antibody is    an immobilizing substance, comprising the steps of:-   (i) adding a required amount of the labeling substance of the    labeled single chain antibody prepared in (i) or (ii) of the    preceding (1) onto a reaction plate compartmentalized into a    plurality of regions having a substance (adapter substance) of (2)    that binds specifically with the labeling substance of the labeled    single chain antibody on the surface thereof, whereby to contact;-   (ii) removing a labeled single chain antibody that was not    immobilized to the substance (adapter substance) that binds    specifically to the labeled single chain antibody on the reaction    plate in the preceding (i); and-   (iii) following the preceding step (ii), removing nonspecific    adsorption from the reaction plate as appropriate;-   (4) preparing a labeled single chain antibody in a case where a    labeling substance is a signal substance, comprising the steps of:-   (i) removing nonspecific adsorption from a reaction plate    compartmentalized into a plurality of regions as appropriate; and-   (ii) adding a required amount of the labeling substance of the    labeled single chain antibody prepared in (i) or (ii) of the    above (1) to the reaction plate;-   (5) adding a required amount of a test substance to a reaction plate    according to the above (3) or (4), and analyzing the binding ability    of a labeled single chain antibody with the test substance; and-   (6) based on the binding ability result obtained in the preceding    (5), qualitatively or quantitatively determining the interaction    between the labeled single chain antibody and the test substance.-   27. A reagent kit for measuring an antigen-antibody reaction,    comprising a reagent to be used in the analysis method according to    the preceding 25 or 26.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a view showing the structure of a translation template of thesingle chain antibody of this invention.

FIG. 2 is a photograph of electrophoresis showing the degree of bindingof biotin to a single chain antibody caused by a biotin ligase.

FIG. 3 is a view showing the degree of specific binding to an antigen ofthe labeled single chain antibody of this invention

FIG. 4 is a view showing a curve of association and dissociation of thelabeled single chain antibody of this invention and an antigen.

FIG. 5 is a view showing the degree of binding between an antigen and asingle chain antibody in which biotin was bonded in an area other than alinker part.

FIG. 6 is a view showing the degree of binding to a nickel column of asingle chain antibody having a polyhistidine peptide in a linker part.

DETAILED DESCRIPTION OF THE INVENTION

(1) Single Chain Antibody and Labeled Single Chain Antibody

A single chain antibody used in this invention may be any kind ofsubstance, as long as it is a substance in which a heavy chain and alight chain of an antibody are connected through a linker and which hasactivity for binding with an antigen for which the antibody has specificbinding affinity. Preferably, the substance used is one in which theheavy chain of an antibody is positioned at the N terminus of the singlechain antibody molecule. As an antibody, a monoclonal antibody havingactivity that recognizes and binds with a specific antigen ispreferable. Further, with respect to a heavy chain and light chain of anantibody, it is not necessary that the substance comprise the fulllength thereof, as long as the substance comprises a part that issufficient for recognizing an antigen and for having specific bindingaffinity thereto. More specifically, a variable region is preferablyused.

A linker is not particularly limited, as long as it has a length that issufficient for a heavy chain and a light chain of an antibody to becrosslinked through the linker, and also has a structure for having alabeling substance. In general, a polypeptide comprising 10 to 30 aminoacids is preferably used. A specific structure can be suitably selectedin accordance with a labeling substance that is described hereunder.

As a labeling substance, a substance that can be used for the purpose oflabelling the single chain antibody of this invention (hereunder, thisis sometimes referred to as “signal substance”) and a substance that canbe used for the purpose of immobilizing the single chain antibody ofthis invention (hereunder, this is sometimes referred to as“immobilizing substance”) are preferable. More specifically, examples ofa signal substance include a fluorescent dye that is capable of bindingto an amino acid, such as a dye belonging to fluorescein, rhodamine,eosin, or NBD; a photosensitizer, such as methylene blue or rose bengal;or a substance that imparts a specific signal in nuclear magneticresonance (NMR), for example an amino acid comprising a fluorine orphosphorus atom. As an immobilizing substance (hereunder, this issometimes referred to as “adapter substance”), any substance may be usedas long as it is a substance that binds with a specific substance thathas been bound to a solid phase surface. Examples of a combination of animmobilizing substance and an adapter substance include various types ofreceptor proteins and a ligand thereof, such as biotin and abiotin-binding protein such as avidin or streptavidin; maltose and amaltose-binding protein; guanine nucleotide and G protein; apolyhistidine peptide and a metal ion such as nickel or cobalt;glutathione-S-transferase and glutathione; a DNA-binding protein and aDNA; an antibody and an antigen molecule (epitope); calmodulin and acalmodulin-binding peptide; ATP-binding protein and ATP; or estradiolreceptor protein and estradiol. Either of these substances may theimmobilizing substance or the adapter substance. Among them, preferablybiotin is used as an immobilizing substance and streptavidin as anadapter substance, or a polyhistidine peptide is used as an immobilizingsubstance and nickel or the like is used as an adapter substance.

A substance that is capable of binding to a polypeptide of a linker partof an antibody in the presence of a specific enzyme in a method forbinding a substance to the linker part can also be used as a labelingsubstance. Examples of this type of substance include biotin and thelike. When using biotin as a labeling substance, examples of a specificenzyme include a biotin ligase, and examples of a linker include asubstance having an amino acid sequence that can be recognized by abiotin ligase.

Further, a labeling substance may be a substance that is incorporated asone part of a linker part of an antibody, and as a specific examplethereof a polyhistidine peptide may be mentioned. In this case, asubstance comprising a polyhistidine peptide may be used as a linker.

Binding of a labeling substance to a linker part, or incorporation alabeling substance therein, can be carried out according to a knownmethod in accordance with the signal substance to be used or theproperties of the immobilizing substance and adapter substance.

(2) Method for Producing Single Chain Antibody and Labeled Single ChainAntibody

A single chain antibody and labeled single chain antibody of thisinvention can be produced, for example, according to the methodsdescribed below. First, (i) a monoclonal antibody that recognizes aprotein of interest or a part thereof as an antigen is prepared, and(ii) DNA encoding the monoclonal antibody is acquired. Then, thesequences encoding the heavy chain and light chain thereof areidentified, and these are linked together sandwiching a nucleotidesequence encoding the linker (hereunder, this DNA fragment may sometimesbe referred to as “single chain antibody unit”). (iii) The protein thatis encoded by the thus-produced single chain antibody unit is thensynthesized by a suitable method that properly maintains the structurethereof. In the case of binding a labeling substance to the linker partat the time of synthesis or after synthesis, the appropriate bindingprocedure is conducted. These methods are described in detail hereunder.

(i) Preparation of Monoclonal Antibody

An antigen of the single chain antibody of this invention is notparticularly limited, and may be any substance as long as the substancehas immunogenicity. More specifically, for example, a sugar chain ofSalmonella or the like may be mentioned. A known method conventionallyused in the art can be used as a method of preparing a monoclonalantibody that specifically recognizes these antigens, and for apolypeptide used as an antigen, a sequence that is suitable as anepitope (antigenic determinant) with high antigenicity can be selectedin accordance with a known method and used. As a method of selecting anepitope, for example, commercially available software such as EpitopeAdviser (manufactured by Fujitsu Kyushu System Engineering) or the likecan be used.

As a polypeptide used as the aforementioned antigen, a synthetic peptidethat was synthesized in accordance with a known method is preferablyused. Although a polypeptide to be used as an antigen may be prepared inan appropriate solution or the like in accordance with a known methodand then used to immunize a mammal such as rabbit or mouse, in order toconduct stable immunization and raise the antibody titer, immunizationis preferably conducted using an antigen peptide that forms a conjugatewith a suitable carrier protein, with the addition of an adjuvant or thelike.

The route of administration of an antigen at the time of immunization isnot particularly limited, and for example, a subcutaneous,intraperitoneal, intravenous or intramuscular route may be used. Morespecifically, for example, a method may be used in which BALB/c mice areinoculated with an antigen polypeptide several times at intervals ofseveral days to several weeks. Regarding intake of the antigen, while anintake of from 0.3 to 0.5 mg/per inoculation is preferable when theantigen is a polypeptide, the intake can be appropriately adjusted inaccordance with the kind of polypeptide and the species of animal to beimmunized.

After immunization, blood is tentatively collected as appropriate toverify an increase in antibody titer by a method such as enzyme-linkedimmunosorbent assay (hereunder, this is sometimes referred to as“ELISA”) or Western blotting, and blood is then collected from an animalin which the antibody titer has increased sufficiently. By subjectingthe obtained blood to a suitable process used in preparation of anantibody, a polyclonal antibody can be obtained. More specifically, forexample, a method may be mentioned in which purified antibody isacquired by purifying the antibody component from serum in accordancewith a known method. A monoclonal antibody can also be produced using ahybridoma produced by fusing myeloma cell and spleen cell of the animalin accordance with a known method (Milstein et al., Nature, 256, 495(1975)). A monoclonal antibody can be acquired, for example, by themethod described below.

First, antibody-forming cells are acquired from the aforementionedanimal in which the antibody titer was raised by immunization of anantigen. Antibody-forming cells are the plasma cells and the precursorcells thereof, lymphoid cells, and while they may be acquired from anypart of the individual, they are preferably acquired from the spleen,lymph node, peripheral blood or the like. As myeloma cells to be fusedwith these cells, in general, an established cell line acquired frommouse, such as 8-azaguanine resistant mouse (derived from BALB/c or thelike) myeloma cell line P3X63-Ag 8.653 (ATCC: CRL-1580) or P3-NS1/1Ag4.1 (Riken Cell Bank: RCB0095) or the like are preferably used. Fusionof the cells can be carried out by mixing the antibody-forming cells andmyeloma cells at an appropriate ratio using a suitable cell fusionmedium, for example, RPMI 1640 or Iscove's modification of Dulbecco'smedium (IMDM), or a medium in which 50% polyethyleneglycol is dissolvedin Dulbecco's modified Eagle's medium (DMEM) or the like. Fusion ofcells can also 10 be conducted by an electrofusion method (U. Zimmermannet al., Naturwissenschaften, 68, 577 (1981)).

A hybridoma can be selected by utilizing the fact that the myeloma cellline used is an 8-azaguanine resistant line and culturing for anappropriate time at 37° C. with 5% CO₂ in a normal culture mediumcontaining a suitable amount of hypoxanthine amino-pterin thymidine(HAT) solution (HAT culture medium). The selection method can besuitably selected and used in accordance with the myeloma cell lineused. A monoclonal antibody can be obtained by analyzing according tothe aforementioned method antibody titers of antibodies produced byselected hybridomas, isolating a hybridoma producing an antibody havinga high antibody titer by a limiting dilution method or the like, andpurifying the monoclonal antibody from culture supernatant obtained byculturing the isolated fused cell in a suitable medium, by anappropriate method such as ammonium sulfate fractionation or affinitychromatography. A commercially available monoclonal antibodypurification kit can also be used to purify the monoclonal antibody.Further, peritoneal fluid containing a large quantity of the monoclonalantibody of this invention can also be obtained by allowing theantibody-producing hybridoma obtained in the manner described above toproliferate intraperitoneally in an animal of the same family as theimmunized animal or in nude mice or the like.

(ii) Acquisition of DNAs Encoding Heavy Chain and Light Chain ofMonoclonal Antibody and Preparation of Single Chain Antibody Unit

As a specific method for acquiring DNAs encoding the heavy chain (Hchain) and the light chain (L chain) of the monoclonal antibody acquiredin the above (i), a method may be mentioned in which the amino acidsequences of one part of the L chain and H chain of immunoglobulinobtained from a hybridoma producing the monoclonal antibody, preferably,parts of the amino acid sequences having a variable region (V region),are analyzed, and on the basis of the amino acid sequences the geneencoding it is cloned. Here, a variable region of the L chain and the Hchain of the monoclonal antibody is preferably a region comprising aframework region (FR) and a hypervariable region (CDR).

As DNA encoding a variable region of a H chain and chain obtained inthis manner, for example, DNA comprising the sequence described inAnand, N. N., et al., J. Biol. Chem., 266, 21874-21879 (1991) and thelike may be mentioned as DNA of a single chain antibody that recognizesO-antigen of Salmonella.

A single chain antibody unit can be prepared by sandwiching DNA encodinga linker between the thus-obtained DNAs encoding the variable regions ofthe H chain and L chain, and connecting the two DNA fragments by anappropriate method. In this case, it is not necessary to obtain thesingle chain antibody unit separately as a DNA fragment, and it may beconstructed at the same time as insertion into an expression vector orthe like as described later. As DNA encoding a linker, any substance maybe used as long as it is DNA encoding a linker as described in (1). Morespecifically, for example, DNA encoding a linker that includes an aminoacid sequence that is recognized by a biotin ligase (Peter J. Schatz(1993), Biotechnology, 11 (1138-1143)) is preferable, and examplesthereof include the substance represented by SEQ ID NO: 1. Further, asan example in which a labeling substance is incorporated as one part ofthe linker part, a substance comprising a nucleotide sequence encoding apolyhistidine peptide or the like may be mentioned.

DNA encoding a linker can be produced using a method usedconventionally, and the DNA is preferably produced by chemicalsynthesis.

(iii) Production of Single Chain Antibody

A single chain antibody can be produced by connecting the thus-obtainedsingle chain antibody unit to a suitable promoter to be under thecontrol thereof, and introducing this into a host, or by conductingtranscription by an appropriate method and then expressing the singlechain antibody under conditions which retain a disulfide bond of thesingle chain antibody to be produced using a cell-free proteintranslation system. An antibody having low binding ability against anantigen can also be acquired as an antibody having higher bindingability by use of a known evolutionary engineering technique.

A suitable promoter can be appropriately selected in accordance with ahost to be used or the RNA synthetase used in transcription. Morespecifically, when using SP6 RNA synthetase for transcription, SP6promoter is preferably used. In the cell-free protein translationsystem, a base sequence that augments translational activity ispreferably inserted between the promoter and the single chain antibodyunit. Specific examples of known base sequences that augmenttranslational activity include the 5′-cap structure (Shatkin, Cell, 9,645- (1976)), Kozak sequence (Kizak, Nucleic Acid Res., 12, 857-(1984))and the like in eucaryotes, and Shine-Dalgarno sequence and the like inprokaryotes. Further, it has been found that translation promotingactivity is also present in the 5′-nontranslated leader sequences of RNAvirus (Japanese Patent No. 2814433), and a method has been developedwhich efficiently conducts protein synthesis using these sequences(Japanese Patent Laid-Open No. 10-146197). In addition, with respect toa random sequence, a sequence obtained by a method that selects atranslation enhancer sequence by taking influence on polysome formationas an indicator may also be mentioned (specification of Japanese PatentApplication No. 2001-396941). Hereunder, DNA produced in this manner maysometimes be referred to as “translation template.”

As a specific example of a translation template, a substance having thestructure shown in FIG. 1 may be mentioned as an example of a substancerecognizing O-antigen of Salmonella.

As a host into which a translation template is introduced, a wheatembryo-derived cell-free protein synthesis system that can be used innormal protein synthesis and which is capable of retaining a disulfidebond of a single chain antibody is used. The reason this system is usedis that an antibody produced with a different cell-free proteinsynthesis system is unable to sufficiently retain a tertiary structurefor recognizing an antigen, and exhibits only a low Kd value (AlexanderZdanov et al., Proc. Natl. Acad. Sci. USA, Vol 91, pp. 6423-6427(1994);C. Roger Mackenzie et al., The Journal of Biological Chemistry, Vol.271, pp. 1527-1533 (1998)). As a specific example of cell extract fromwheat embryo to be used in this invention, the commercially availableProteios™ (manufactured by Toyobo Co., Ltd.) or the like may bementioned.

Further, a reaction solution which can retain an intramoleculardisulfide bond and also synthesize a protein can be prepared byadjusting the concentration of a reducing agent among the ingredientsnecessary for protein synthesis of a reaction solution of the abovewheat embryo-derived cell-free translation system (hereunder, this issometimes referred to as “weak reductive translation reactionsolution”). Examples of a specific reducing agent and the concentrationthereof include dithiothreitol (hereunder, sometimes referred to as“DTT”) at a final concentration of 20 to 70 μM, preferably 30 to 50 μM,2-mercaptoethanol at a final concentration of 0.1 to 0.2 mM, andglutathione/oxidized glutathione at a final concentration within a rangeof 30 to 50 μM/1 to 5 μM.

The concentration of a reducing agent in the translation reactionsolution is not limited to the above concentrations, and may be suitablymodified in accordance with the protein to be synthesized. While amethod for selecting the range of optimal concentration of a reducingagent is not particularly limited, for example, a method in whichassessment is made based on the effect of an enzyme catalyzing adisulfide bond exchange reaction may be mentioned. More specifically,translation reaction solutions in which the concentration of a reducingagent is variously adjusted are prepared, and an enzyme that catalyzes adisulfide bond exchange reaction is added to these solutions, to conductsynthesis of a protein having an intramolecular disulfide bond. As acontrol experiment, protein synthesis is carried out in a similar mannerin the same translation reaction solutions without adding the enzymethat catalyzes a disulfide bond exchange reaction. A solubilizedcomponent of the protein synthesized in the above manner is thenisolated by, for example, a method such as centrifugation. A reactionsolution in which the solubilized component constitutes 50%(solubilization ratio 50%) or more of the total volume and in which thesolubilized component increased after addition of the enzyme thatcatalyzes a disulfide bond exchange reaction can be judged as suitableas a reaction solution that conducts synthesis while retaining anintramolecular disulfide bond of the protein in its original state.Further, of the ranges of concentration of a reducing agent that wasselected on the basis of the aforementioned effect of an enzyme thatcatalyzes a disulfide bond exchange reaction, concentrations of areducing agent that generate the largest amount of synthesized proteincan be selected as a further preferable concentration range.

Methods that can be used to prepare a reaction solution having theaforementioned reducing agent concentration include a method in whichcell extract for wheat embryo-derived cell-free protein synthesis thatdoes not include a reducing agent is prepared, and then ingredientsrequired for a wheat embryo-derived cell-free protein translation systemare added thereto together with a reducing agent at a concentrationwithin the above concentration range, and a method in which a reducingagent is removed from cell extract for wheat embryo-derived cell-freeprotein synthesis such that the concentration of the reducing agent iswithin the aforementioned concentration range. Since cell extract forwheat embryo-derived cell-free protein synthesis requires advancedreducing conditions when extracting, a method in which a reducing agentis removed from this solution after extraction is more convenient. As amethod for removing a reducing agent from cell extract, a method using acarrier for gel filtration and the like may be mentioned. Morespecifically, for example, a method in which a Sephadex G-25 column ispreviously equilibrated with a suitable buffer solution that does notinclude a reducing agent, and cell extract is then passed therethroughmay be mentioned.

Further, the cell extract may also be used after forming the cellextract into a lyophilized product by lyophilizing, and adding asuitable buffer solution thereto. Preferably the total concentration ofa deliquescent substance is made 60 mM or less when lyophilizing.Lyophillization can also be conducted after adding the aforementionedtranslation template to the cell extract.

Further, for a substance exhibiting deliquescence (deliquescentsubstance) in the aforementioned lyophilized product, a content thatdoes not lower storage stability in a lyophilized condition ispreferably 0.01 parts by weight or less relative to 1 part by weight ofa protein contained in the lyophilized product, and particularlypreferably the content is 0.005 parts by weight or less relativethereto. In this connection, the weight of a protein mentioned hererefers to a weight calculated by measurement of absorbance (260, 280,320 nm).

Hereunder, cell extract in which the concentration of a reducing agentwas adjusted as described above is sometimes referred to as “weakreductive translation reaction solution.”

Further, by carrying out a translation reaction in which an enzyme thatcatalyzes a disulfide bond exchange reaction is further added to a weakreductive translation reaction solution, it is possible to conducthighly efficient synthesis of a protein that retains an intramoleculardisulfide bond. As an enzyme that catalyzes a disulfide bond exchangereaction, for example, protein disulfide isomerase or the like may bementioned. The amount of these enzymes to be added to a wheatembryo-derived cell-free translation system can be suitably selected inaccordance with the kind of enzyme. More specifically, when addingprotein disulfide isomerase to a translation reaction solution that iscell extract for cell-free protein synthesis extracted from wheatembryo, which contains as a reducing agent 20 to 70 μM of DTT, andpreferably 30 to 50 μM thereof, the protein disulfide isomerase is addedto bring to a final concentration within the range of 0.01 to 10 μM, andpreferably 0.5 μM. With respect to the stage for adding an enzyme, fromthe viewpoint of efficiency of disulfide bond formation, the enzyme ispreferably added prior to the start of the cell-free translationreaction.

Examples of cell-free protein translation systems derived from seed ofplants other than wheat include those derived from gramineous plantssuch as barley, rice and corn. However, of these cell-free proteintranslation systems, use of wheat embryo extract is particularlypreferable, and a method for producing a single chain antibody will beexplained in detail below taking as an example a case using this cellextract.

As a method for selecting wheat embryo, for example, the method ofJohnston, F. B., et al., Nature, 179, 160-161 (1957) can be used, and asa method for producing cell extract from the embryo, a method describedin Erickson, A. H., et al. Meth. In Enzymol., 96, 38-50 (1996) or thelike can be used.

According to a preparation method advantageously utilized in thisinvention, wheat embryo extract can be obtained by collecting wheatembryo extract and purifying the extract by gel filtration or the like.Gel filtration can be conducted, for example, using a gel filtrationdevice such as a Sephadex G-25 column. The compositions andconcentrations of the various components in a gel filtration solutionare known in the art, and those used in a method for producing wheatembryo extract for cell-free protein synthesis may be adopted. Withrespect to a solution for equilibrating a Sephadex G-25 column, by usinga solution that does not contain a reducing agent, more specifically,for example, a solution containing HEPES-KOH, potassium acetate,magnesium acetate, or L-form amino acids, approximately 97% of areducing agent contained in the extract can be absorbed. Specifically,when extraction is conducted using extract from wheat embryo containing1 mM of DTT as a reducing agent, it is possible to ultimately obtainwheat embryo extract containing approximately 30 μM of DTT. However,because the activity of wheat embryo extract in which the concentrationof a reducing agent has been lowered is noticeably reduced bycryopreservation, a step of removing a reducing agent is preferablyconducted immediately prior to a translation reaction in which theextract is to be used.

Microorganisms, in particular spores such as filamentous bacteria (mold)may sometimes be contained in the embryo extract after gel filtration,and these microorganisms are preferably removed. Since proliferation ofmicroorganisms is observed, in particular, in long-term (1 day or more)cell-free protein synthesis reaction, the prevention thereof isimportant. Although a technique for removing microorganisms is notparticularly limited, the use of a filtration sterilization filter ispreferable. The pore size of a filter is not particularly limited aslong as the filter is capable of removing microorganisms that maycontaminate the extract, and normally a pore size of 0.1 to 1micrometer, preferably 0.2 to 0.5 micrometers, is adequate. In thisconnection, since the size of small categories of spores of Bacillussubtilis is 0.5 μm×1 μm, the use of a 0.20 micrometer filter (forexample, Minisart™, manufactured by Sartorius HPLC) is also effectivefor removing spores. When filtering, preferably, filtering is firstconducted using a filter with a large pore size, and then filtering isconducted using a filter with a pore size that is capable of removingmicroorganisms that may be contained in the initial filtrate.

Cell extract obtained in this manner is purified to the extent thatendosperm containing a substance that inhibits a protein synthesisfunction (a substance such as tritin, thionin or a ribonuclease thatacts on mRNA, tRNA, a translation protein factor or ribosome or the likeand inhibits the function thereof) retained by or contained by thesource cell itself is almost completely removed. Here, the term“purified to the extent that endosperm is almost completely removed”refers to wheat embryo extract in which an endosperm part is removed tothe extent that ribosome is not substantially deadenylated, and the term“extent that ribosome is not substantially deadenylated” refers to thedeadenylation rate of ribosome being less than 7%, and preferably 1% orless.

Further, since this type of cell extract from which an endospermcomponent has been removed contains low-molecular protein synthesisinhibitors (hereunder, these may be referred to as “low-molecularsynthesis inhibitors”), preferably these low-molecular synthesisinhibitors are removed by fractionation from the components of the cellextract utilizing difference in molecular weight. It is sufficient thatthe molecular weight of substances to be removed (low-molecularinhibitors) be lower than that of factors necessary for proteinsynthesis contained in the cell extract. More specifically, substanceshaving a molecular weight of 50,000 to 14,000 or less, and preferably14,000 or less may be mentioned.

As a method for removing low-molecular synthesis inhibitors from cellextract, a known method conventionally used in the art can be used, andas a specific example thereof a method using dialysis through a dialysismembrane, gel filtration, or ultrafiltration may be mentioned. Of these,a method using dialysis (dialysis method) is preferable from theviewpoint of ease of supply of the substance to an internal dialysissolution. Hereunder, an example when using a dialysis method isdescribed in detail.

As a dialysis membrane to be used in the dialysis, a membrane with amolecular weight cutoff of 50,000 to 12,000 may be mentioned, and morespecifically, a regenerated cellulose membrane with a molecular weightcutoff of 12,000 to 14,000 (manufactured by Viskase Sales, Chicago) orSpectraPor 6 with a molecular weight cutoff of 50,000 (manufactured bySpectrum Laboratories Inc., Ca., USA) or the like may be used. Dialysisis conducted according to a conventional method by introducing asuitable volume of the above-described cell extract into this type ofdialysis membrane. A time period for conducting dialysis is preferablybetween around 30 minutes and 24 hours.

In a case where an insoluble component is generated in cell extract whenremoving low-molecular synthesis inhibitors, it is possible to enhancethe protein-synthesizing activity of the ultimately obtained cellextract (hereunder, this is sometimes referred to as “post-treatmentcell extract”) by inhibiting the generation of this insoluble component(hereunder, this is sometimes referred to as “stabilization of cellextract”) As a specific method for stabilizing the cell extract, amethod in which removal of the aforementioned low-molecular inhibitorsis conducted in a solution containing at least a high-energy phosphatecompound such as ATP or GTP may be mentioned. As a high-energy phosphatecompound, ATP is preferably used. Removal is preferably conducted in asolution containing ATP and GTP, and further preferably, ATP, GTP and 20kinds of amino acid.

When conducting removal of low-molecular inhibitors in a solutioncontaining these components (hereunder, sometimes referred to as“stabilization components”), the step of removing the low-molecularinhibitors may be conducted after previously adding the stabilizationcomponents to the cell extract and incubating the solution. When using adialysis method to remove low-molecular synthesis inhibitors, removal ofthe low-molecular inhibitors can be conducted by adding stabilizationcomponents to not only the cell extract but also to an external dialysissolution to conduct dialysis. Adding stabilization components to theexternal dialysis solution enables the constant supply of newstabilization components even though stabilization components aredegraded during dialysis, and is thus more preferable. This techniquecan also be applied when using gel filtration or ultrafiltration, inwhich case a similar effect can be obtained by first equilibrating therespective carriers using a buffer for filtration containingstabilization components, and then providing the cell extract containingstabilization components for filtration and conducting the filtrationwhile adding the aforementioned buffer.

The added amount of stabilization components and the time forstabilization treatment can be suitably selected in accordance with thetype of cell extract and method of preparation. As an example of amethod for selecting these, a method may be mentioned in which tentativeamounts and kinds of stabilization components are added to cellextracts, a step of removing low-molecular inhibitors is then conductedafter an appropriate period, the obtained post-treatment cell extractsare separated into solubilized components and insolubilized componentsby a method such as centrifugation, and of these the cell extract whichhas less insolubilized components is selected. Another preferable methodis one in which cell-free protein synthesis is conducted using theobtained post-treatment cell extracts, and of these the cell extractwith the highest protein-synthesizing activity is selected. When usingcell extract with a dialysis method in the above selection methods, amethod may be mentioned in which suitable stabilization components arealso added to external dialysis solutions, and after conducting dialysisfor an appropriate period using these, selection is conducted based onthe amount of insoluble substances in the obtained cell extracts, theprotein-synthesizing activity of the obtained cell extracts, or thelike.

As a specific example of stabilization conditions for cell extractselected in this manner, for a case of conducting a step of removinglow-molecular inhibitors by a dialysis method with the aforementionedprepared wheat embryo extract, a method may be mentioned in which 100 μMto 0.5 mM of ATP, 25 μM to 1 mM of GTP, and 25 μM to 5 mM each of 20kinds of L-form amino acids are added to the external dialysis solutionand wheat embryo extract and dialysis is conducted for 30 minutes to 1hour or more. Dialysis may be conducted at any temperature, as long asthe temperature is one at which protein synthesizing activity is notlost and dialysis is possible. More specifically, the lowest temperatureis one at which the solution does not freeze, and this is normally −10°C., preferably −5° C., and the highest temperature is 40° C., which isthe limit for a temperature that does not impart an adverse effect on asolution used in dialysis, and 38° C. is preferable.

A method for adding stabilization components to cell extract is notparticularly limited, and a method may be employed in whichstabilization components are added to cell extract prior to a step ofremoving low-molecular inhibitors, the resulting mixture is incubatedfor a suitable period to undergo stabilization, and then a step ofremoving low-molecular synthesis inhibitors is performed, or a methodmay be employed in which a step of removing low-molecular synthesisinhibitors is performed using cell extract to which stabilizationcomponents were added and/or buffer solution for use in the removal stepto which stabilization components were added.

Protein synthesis can be performed by preparing the aforementioned cellextract for cell-free protein synthesis with a concentration of areducing agent that is within the above-described ranges, addingthereto, as necessary, an energy source or amino acids, translationtemplate or tRNA or the like that are required for cell-free proteinsynthesis, as well as an enzyme that catalyzes a disulfide bond exchangereaction, and introducing the resulting mixture into respectivelyselected known systems or apparatuses. Examples of a system or apparatusfor protein synthesis include a batch method (Pratt, J. M., et al.,Transcription and Translation, 179-209, Hames, B. D. & Higgins, S. J.,Eds., IRL Press, Oxford (1984)), a continuous cell-free proteinsynthesis system that continuously supplies amino acids, energy sourcesand the like to a reaction system (Spirin, A. S., et al., Science, 242,1162-1164 (1988)), a dialysis method (Kigawa, et al., 21st AnnualMeeting of the Molecular Biology Society of Japan, WID 6), and a overlaymethod (Sawasaki, T., et al., FEBS Let., 514, 102-105 (2002)).

In addition, a method which supplies template RNA, amino acids, anenergy source or the like to a synthesis reaction system when requiredand which removes a synthesized product or degradation product at arequired time (Japanese Patent Laid-Open No. 2000-333673; hereunder,this is sometimes referred to as “discontinuous gel filtration method”)or the like can be used.

Among these, while use of a system that supplies amino acids or anenergy source continuously or discontinuously allows a reaction to bemaintained for a long period and thereby enables greater efficiency, useof a batch method is preferable when conducting protein synthesis usinga weak reductive translation reaction solution, as the protein synthesisefficiency tends to be high. Further, when preparing wheat embryoextract by the method described above, the addition of tRNA is normallynot necessary, as a sufficient amount of tRNA is already containedtherein.

When conducting protein synthesis by a batch method, the protein can besynthesized, for example, by preincubating a synthesis reaction solutionfrom which a translation template has been excluded for a suitableperiod as necessary, and then adding the translation template andincubating. As a synthesis reaction solution, for example, a solutionthat contains 10 to 50 mM of HEPES-KOH (pH 7.8), 55 to 120 mM ofpotassium acetate, 1 to 5 mM of magnesium acetate, 0.1 to 0.6 mM ofspermidine, L-form amino acids (0.025 to 1 mM each), 20 to 70 μM,preferably 30 to 50 μM of DTT, 1 to 1.5 mM of ATP, 0.2 to 0.5 mM of GTP,10 to 20 mM of creatine phosphate, 0.5 to 1.0 U/μl of RNase inhibitor,0.01 to 10 μM of protein disulfide isomerase and 24 to 75% of wheatembryo extract can be used as a translation reaction solution.

When using this kind of translation reaction solution, preincubation isconducted at 10 to 40° C. for 5 to 10 minutes, and incubation issimilarly conducted at 10 to 40° C., preferably, 18 to 30° C., andfurther preferably at 20 to 26° C. A reaction time is the time untilreaction stops, and in the batch method this is normally from about 10minutes to 7 hours (see Pratt, J. M., et al., Transcription andTranslation, 179-209, Hames, B. D. & Higgins, S. J., Eds., IRL Press,Oxford (1984)).

When conducting protein synthesis by a dialysis method, proteinsynthesis is conducted using an apparatus that conducts separation bymeans of an external dialysis solution and a dialysis membrane thatallows mass transfer, employing the synthesis reaction solution as theinternal dialysis solution (see Kigawa, et al., 21st Annual Meeting ofthe Molecular Biology Society of Japan, WID 6).

When conducting protein synthesis using the overlay method, proteinsynthesis is conducted by inserting the synthesis reaction solution intoa suitable container and then overlaying the external dialysis solutionas described in the above dialysis method on the reaction solution in amanner that does not disturb the interface therebetween (see Sawasaki,T., et al., FEBS Let., 514, 102-105 (2002); International PatentPublication No. WO 02/24939 A1).

When conducting protein synthesis using the discontinuous gel filtrationmethod, protein synthesis is performed by carrying out synthesisreaction using a synthesis reaction solution, and when the synthesisreaction stops, supplying template RNA, amino acids, an energy sourceand the like thereto, and removing a synthesized product or degradationproduct. More specifically, for example, after preincubating asnecessary the aforementioned synthesis reaction solution from which atranslation template has been excluded for an appropriate time, atranslation template is added thereto and the solution is inserted intoa suitable container to undergo reaction. Examples of a containerinclude a microplate or the like. Under this reaction, for example, inthe case of a reaction solution containing 48% part by volume of wheatembryo extract relative to the total volume, the synthesis reaction willstop completely after 1 hour of reaction. This can be confirmed bypolyribosome analysis (Proc. Natl. Acad. Sci. USA, 97, 559-564 (2000))using sucrose density gradient centrifugation or measurement of theincorporation of amino acids into the protein. The above reactionsolution in which synthesis reaction has stopped is passed through a gelfiltration column that was previously equilibrated by a supply fluidhaving a similar composition to the external dialysis solution describedin the above dialysis method. By reheating this filtrate solution to asuitable reaction temperature, synthesis reaction re-starts and proteinsynthesis progresses over several hours. The reaction and gel filtrationoperations are then repeated. A reaction temperature and time can beappropriately selected in accordance with the protein synthesis systemused, and in a system using wheat embryo extract the gel filtration ispreferably repeated every approximately 1 hour at 26 ° C.

When bonding a labeling substance to the single chain antibody of thisinvention in the presence of a specific enzyme according to this kind ofcell-free protein translation, the above-described translation reactionis conducted in the presence of the labeling substance and an enzymethat is capable of binding the labeling substance to a polypeptide of alinker part. More specifically, when bonding biotin as a labelingsubstance to a linker, a translation reaction is conducted in thepresence of, for example, a biotin ligase (Avidity, manufactured by LLC,or the like) that is an enzyme that bonds biotin by recognizing an aminoacid recognized by a biotin ligase that is previously inserted into alinker. An added amount of biotin and the biotin ligase is preferably anamount described in the instructions accompanying a commerciallyavailable product (enzyme).

When bonding labeling substances after synthesis of the protein, aftercompleting the translation reaction bonding may be conducted to a linkerpart of the single chain antibody in the translation reaction solutionby a method suitable for the respective labeling substances, or bondingmay be conducted by a method suitable for the respective labelingsubstances after purifying the single chain antibody by the methoddescribed below.

The thus obtained single chain antibody or labeled single chain antibodyof this invention can be confirmed by a known method. More specifically,for example, a method involving measuring incorporation of amino acidinto protein, separation by SDS-polyacrylamide gel electrophoresis andstaining by Coomassie brilliant blue (CBB), or autoradiography (Endo,Y., et al., J. Biotech., 25, 221-230 (1992); Proc. Natl. Acad. Sci. USA,97, 559-564 (2000)) or the like can be used.

Further, since the single chain antibody or labeled single chainantibody of interest is contained at a high concentration in thethus-obtained reaction solution, the single chain antibody or labeledsingle chain antibody of interest can be easily acquired from thereaction solution by a known method of separation and purification, suchas dialysis, ion-exchange chromatography, affinity chromatography or gelfiltration.

(3) Utilization of Labeled Single Chain Antibody

The labeled single chain antibody of this invention can be used in amethod for analyzing an antigen-antibody reaction by analyzing thebinding ability thereof against an antigen. A method for analyzing anantigen-antibody reaction can be performed by comprising the followingsteps (I) to (VI):

-   (I) preparing a labeled single chain antibody under conditions in    which a disulfide bond of a single chain antibody is retained,    comprising the step of the following (1) or (2):-   (1) producing a labeled single chain antibody by subjecting a DNA,    in which DNAs encoding a heavy chain and a light chain of an    antibody having binding ability with a specific antigen are linked    through a DNA encoding a linker comprising a nucleotide sequence    that is capable of binding with a labeling substance in the presence    of a specific enzyme after translation, to transcription and    translation using a wheat cell-free protein synthesis system in the    presence of a specific enzyme; or-   (2) producing a labeled single chain antibody by subjecting a DNA,    in which DNAs encoding a heavy chain and a light chain that are    variable regions of an antibody having binding ability with a    specific antigen are linked through a DNA encoding a linker    comprising a nucleotide sequence that is capable of binding with a    labeling substance in the presence of a specific enzyme after    translation, to transcription and translation using a wheat    cell-free protein synthesis system in the presence of a specific    enzyme;-   (II) preparing a substance (adapter substance) that binds    specifically with a labeling substance of a labeled single chain    antibody in a case where the labeling substance of the labeled    single chain antibody is an immobilizing substance, comprising the    steps of:-   (1) immobilizing a substance (adapter substance) that binds    specifically with a labeling substance of a labeled single chain    antibody on a reaction plate compartmentalized into a plurality of    regions;-   (2) removing the substance (adapter substance) that binds    specifically with a labeling substance of a labeled single chain    antibody~that was not immobilized to the reaction late in the    preceding (i); and-   (3) before and after the step of the preceding (i) or (ii), removing    nonspecific adsorption from the reaction plate as appropriate;-   (III) preparing an immobilized single chain antibody in a case where    a labeling substance is an immobilizing substance, comprising the    steps of:-   (1) adding a required amount of the labeled single chain antibody    prepared in (1) or (2) of the above (I) to a reaction plate    compartmentalized into a plurality of regions having a substance    (adapter substance) that binds specifically with the labeled single    chain antibody of the above (II) on the surface thereof, to contact    the labeled single chain antibody with the adapter substance;-   (2) removing a labeled single chain antibody that was not    immobilized to the substance (adapter substance) that binds    specifically to the labeled single chain antibody on the reaction    plate in the preceding (1); and-   (3) following the preceding step (2), removing nonspecific    adsorption from the reaction plate as appropriate;-   (IV) preparing a labeled single chain antibody in a case where a    labeling substance is a signal substance, comprising the steps of:-   (1) removing nonspecific adsorption from the reaction plate    compartmentalized into a plurality of regions as appropriate; and-   (2) adding a required amount of the labeling substance of the    labeled single chain antibody prepared in (1) or (2) of the    above (I) to the reaction plate;-   (V) adding a required amount of a test substance to each reaction    plate according to the above (III) or (IV), and analyzing the    binding ability of the labeled single chain antibody with the test    substance; and-   (VI) based on the binding ability result obtained in the preceding    (V), qualitatively or quantitatively determining the interaction    between the labeled single chain antibody and the test substance.

In the above antigen-antibody analysis method, conditions whereby adisulfide bond of a single chain antibody is retained are notparticularly limited, as long as the conditions enable the retention ofa disulfide bond of a labeled single chain antibody produced in a stepof producing a labeled single chain antibody. More specifically, themethod described in (iii) production of single chain antibody, which canbe carried out by adjusting the concentration of a reducing agent in atranslation reaction solution may be mentioned. Further, a method forremoving an adapter substance and labeled single chain antibodycomprises removing the adapter substance and labeled single chainantibody from the top of a reaction plate by washing the reaction plateseveral times using a washing buffer normally used by those skilled inthe art. A method for removing nonspecific adsorption from an adaptersubstance immobilized on a reaction plate refers to using a blockingsolution or the like that is conventionally used by those skilled in theart to fill the reaction plate. Thereafter, washing can be conductedseveral times with a buffer solution.

When a single chain antibody was immobilized, a method that is known inthe art can be used to reduce nonspecific adsorption. Specific examplesthereof include a method of precoating an array solid support usingbovine serum albumin (BSA), reduced low fat milk, salmon sperm DNA, pig(mucosal) heparin or the like (Ausubel et al., Short Protocols inMolecular Biology, 3rd edition (1995)).

As a reaction plate used in the aforementioned antigen-antibody analysismethod, a reaction plate that is suitable for an apparatus or method foranalyzing an antigen-antibody reaction can be used. More specifically,when conducting analysis by enzyme-linked immunosorbent assay (ELISA)(Crowthjer, J. R., Methods in Molecular Biology, 42, (1995)), a plasticmicrotiter plate that is normally used in the ELISA method is preferred.When using a surface plasmon resonance method (Cullen, D. C., et al.,Biosciences, 3(4), 211-225 (1987-88)), a reaction plate in which ametallic thin film of gold, silver, platinum or the like is formed on atransparent reaction plate made of glass or the like is preferred.Further, when using molecule imaging using an evanescent field (Funatsu,T., et al., Nature, 374, 555-559 (1995)), a transparent medium made ofglass or the like is preferable, and more preferably a reaction platemade of quartz glass is used. When using fluorescent imaging analysis, anylon membrane or nitrocellulose membrane that is normally used forimmobilizing a protein or the like can be used, and a plastic microtiterplate or the like can also be used. Further, a complex carbohydrate (forexample, agarose and sepharose), acrylic resin (for example,polyacrylamide and latex beads), magnetic beads, silicon wafer and thelike can also be used as reaction plates.

Bonding of an adapter substance to this kind of reaction plate can beperformed according to a known method that is conventionally used in theart. More specifically, a diazo process, a peptide process (using acidamide derivatives, carboxychloride resin, maleic anhydride derivatives,isocyanate derivatives, cyanogen bromide activated polysaccharides,cellulose carbonate derivatives or the like), alkylation process, amethod using a crosslinking reagent, a method using Ugi reaction and thelike may be mentioned. When using a reaction plate made of glass or thelike, a method that conducts physical adsorption can also be used.Further, a commercially available product such as streptavidin magneticbeads (manufactured by Promega Corp.) can also be used.

By bringing the thus obtained labeled single chain antibody into contactwith a solution containing one or more test substances such as knownantigens and analyzing the antigen-antibody reaction, it is possible toidentify an antibody having binding specificity with respect to theantigen. The antigen may be a protein, or may be an organic compound,carbohydrate, nucleic acid or the like. These may be isolated, or may berecombinant or naturally occurring substances. The amount of an antigenused herein is preferably in the range of approximately 1 to 100 ng/μl.The time required for an antigen-antibody reaction is normally withinthe range of 5 minutes to 24 hours, and in general a time between 0.5 to2 hours is preferable.

After the antigen-antibody reaction, in the case of an immobilizedsingle chain antibody, a step can be added of washing the solid phase towhich the antibody is bound using a buffer containing surfactants or thelike that can be used biochemically. The composition of the buffer andthe number of washings and the like can be appropriately selected inaccordance with the strength of the antigen-antibody reaction and thelike.

Further, in the aforementioned method for analyzing an antigen-antibodyreaction, the antigen-antibody reaction can be analyzed by analyzing thebinding ability between the immobilized single chain antibody and theantigen when the labelings substance is an immobilizing substance, andby analyzing the with the antigen in a solution when the labelingsubstance is a signal substance.

A method for quantitatively or qualitatively determining interactionbetween a labeled single chain antibody and a test substance can beconducted according to a known method that is conventionally used in theart. More specifically, a method such as ELISA, surface plasmonresonance, molecular imaging utilizing an evanescent field, fluorescentimaging analysis or a method using radioisotope labels may be mentioned.

A test substance such as an antigen may be any substance that maycomprise an antigen. Specific examples thereof include body fluid suchas blood, bacterial cell wall extract, and a protein mixture.

According to the method for analyzing an antigen-antibody reaction usingthe labeled single chain antibody of this invention and a reagent kitfor measuring an antigen-antibody reaction comprising a reagent used inthe analysis method, for example, a tool for diagnosing and analyzingthe presence or absence of a human autoantibody, a cancer cell specificantigen and the like can be provided.

EXAMPLES

This invention is described in further detail hereunder by means ofexamples, however the scope of this invention is not limited by theseexamples.

Example 1 Preparation of Biotinylated anti-Salmonella Single ChainAntibody

(1) Preparation of DNA Encoding Salmonella Single Chain Antibody andLinker

An anti-Salmonella single chain antibody was selected as the singlechain antibody of this invention to conduct the following test. TheX-ray conformation of this antibody has already been analyzed, andmolecular recognition with respect to sugar chain has been investigatedin detail (Cygler, M., et al., Science, 253, 442-445 (1991); Bundle, D.R., et al., Biochemistry, 33, 5172-5182 (1994)). Lipopolysaccharide ispresent on the cell cortex of Salmonella bacteria, and anti-Salmonellaantibody binds to O-antigen that is located at the most extracellulardomain of the lipopolysaccharide (Anand, N. N., et al., Protein Engin.,3, 541-546 (1990)). It has been reported that a single chain antibody inwhich VL chain and VH chain, antigen-recognition sites that bindspecifically to O-antigen, were connected by a specific linker wasexpressed in large quantities using Escherichia coli (Anand, N. N., etal., J. Biol. Chem., 266, 21874-21879 (1991)). Since a formation inwhich one disulfide bond is present respectively in the VL chain and VHchain is indispensable to synthesize a single chain antibody in anactive form (Zdanov, A. L. Y., et al., Proc. Natl. Acad. Sci. USA, 91,6423-6427 (1994)), the aforementioned single chain antibody was used asthe subject of the method of this invention. DNA encodinganti-Salmonella single chain antibody was acquired by conducting apolymerase chain reaction (PCR) employing a plasmid containing DNAencoding a single chain antibody against wild-type Salmonella O-antigen(Anand, N. N., et al., J. Biol. Chem., 266, 21874-21879 (1991)) as atemplate and using primers comprising the nucleotide sequencesrepresented by SEQ ID NOS: 2 and 3. The acquired DNA fragments wereligated into pGEMT-easy Vector (from Promega Corp.), and then digestedwith the restriction enzymes BgIII and NotI. The obtained DNA fragmentswere inserted into pEU vector that had been previously digested with thesame restriction enzymes. PCR was conducted employing this plasmid as atemplate and using primers comprising the nucleotide sequencesrepresented by SEQ ID NOS: 4 and 5 to introduce a stop codon. The thusproduced plasmid was designated “scfv-pEU”.

Next, DNA was produced in which a DNA sequence (SEQ ID NO: 1) encoding abiotin ligase recognition sequence was inserted in a linker part. First,PCR was carried out employing as a template the plasmid scfv-pEUproduced as described above, and using LA Taq (manufactured by TakaraCo., Ltd.) kit with primers comprising the nucleotide sequencesrepresented by SEQ ID NOS: 6 and 7. The PCR reaction solution wasprepared using 5 μl of 10× LA buffer, 5 μl of 25 mM magnesium chloride,8 μl of 2.5 mM dNTP, 1 μl of 20 μM primer (for each primer), and 0.1 ngof template plasmid/50 μl, and reaction was conducted at 94 ° C. for 1min×1 cycle, 94 ° C. for 45 sec/55° C. for 1 min/72° C. for 1.5 min×30cycles, and then 72° C. for 5 min. In accordance with a conventionalmethod, the ends of amplified DNA fragments were blunted using KOD T4polymerase (manufactured by NEB Inc.), the fragments were phosphorylatedwith Polynucleotide Kinase (NEB Inc.), and self-ligation was thencarried out using Ligation High (manufactured by Toyobo Co., Ltd.) toproduce a circular plasmid (FIG. 1; hereafter, this is sometimesreferred to as “scFv-biotin-pEU”).

(2) Preparation of Cell Extract for Weak Reduced Form of Cell-freeProtein Synthesis

Hokkaido-produced Chihoku wheat seeds (unsterilized) were added to amill (Rotor Speed mill pulverisette 14 model, manufactured by FritschInc.) at a rate of 100 g per minute, to gently pulverize the seeds at arotation speed of 8,000 rpm. After sieving to collect fractionscontaining embryo having germinating capacity (mesh size 0.7 to 1.00mm), a floating fraction containing embryo with germinating capacity wascollected by flotation using a mixed solution of carbon tetrachlorideand cyclohexane=(volume ratio=carbon tetrachloride:cyclohexane=2.4:1),the organic solvent was removed by drying at room temperature, andimpurities such as seed coat that were mixed therein were removed byblowing air at room temperature to obtain a coarse embryo fraction.

Next, embryo was selected from the coarse embryo fraction by utilizingdifference in color using a belt type color sorter BLM-300K(manufactured by Anzai Manufacturing Co. Ltd.; selling agent: AnzaiCorporation, Ltd.) in the manner described below. The color sorter is anapparatus having means to irradiate light on a coarse embryo fraction,means to detect reflected light and/or transmitted light from the coarseembryo fraction, means to compare detected values and reference values,and means to select and remove components with a detected value that isoutside the reference values or components with a detected value that iswithin the range of reference values.

Coarse embryo fractions were supplied onto a beige color belt of thecolor sorter to form an amount of 1000 to 5000 grains/cm², light wasirradiated by fluorescent lamp onto the coarse embryo fractions on thebelt, and the reflected light was detected. The conveying speed of thebelt was 50 m/min. A monochrome CCD line sensor (2048 pixels) was usedas a light-receiving sensor.

First, in order to remove black-colored components (seed coat etc.) fromthe embryo, the reference value was set between the brightness of theembryo and the brightness of the seed coat, and components having avalue outside the reference value were removed by suction. Subsequently,in order to screen for endosperm, the reference value was set betweenthe brightness of the embryo and the brightness of endosperm, andcomponents having a value outside the reference value were removed bysuction. Suction was conducted using 30 suction nozzles provided atpositions of about 1 cm apart on the upper part of the conveyor belt(the suction nozzles were arranged in a condition of 1 nozzle per 1 cmlength).

By repeating this method, embryo was screened until the purity of theembryo (weight ratio of embryo contained per 1 g of arbitrary sample)was 98% or more.

The obtained wheat embryo fraction was suspended in distilled water witha temperature of 4° C., and washed using an ultrasonic washer until thecleaning fluid lost its white turbidity. Next, the fraction wassuspended in a solution containing 0.5 % Nonidet P40 (manufactured byNacalai Tesque Inc.), and washed using an ultrasonic washer until thecleaning fluid lost its white turbidity to obtain wheat embryo, afterwhich the following process was conducted at 4° C.

Extracting solvent (80 mM HEPES-KOH (pH 7.8), 200 mM potassium acetate,10 mM magnesium acetate, and 8 mM dithiothreitol (0.6 mM each of 20kinds of L-form amino acid may also be added)) of two-fold volumerelative to the wet weight of the washed embryo was added thereto, andlimited pulverization of the embryo was conducted 3 times for 30 secondseach time at 5,000 to 20,000 rpm using a Waring blender. Centrifugedsupernatant obtained from this homogenate by centrifugation at 30,000×gfor 30 min using a high-speed centrifuge was centrifuged again under thesame conditions to obtain supernatant. A decline in activity was notobserved for this sample after long-term storage at −80° C. or below.The obtained supernatant was filtrated through a filter with a pore sizeof 0.2 μM (New SteraDisk 25; manufactured by Kurabo Industries Ltd.) forfilter sterilization and removal of minute contaminants.

Next, this filtrate was subjected to gel filtration using a SephadexG-25 column that was previously equilibrated with a mixed solution (40nM HEPES-KOH (pH 7.8), 100 mM potassium acetate, 5 mM magnesium acetate,and 0.3 mM each of 20 kinds of L-form amino acids (the amino acids maybe omitted in accordance with the purpose of protein synthesis, orlabeled amino acids may be used). After centrifuging the obtainedfiltrate again at 30,000×g for 30 min and adjusting the concentration ofthe collected supernatant so that A260 nm was 90 to 150 (A260/A280=1.4to 1.6), the supernatant was stored at −80° C. or below until use in theprotein synthesis reaction or dialysis process described hereunder.

(3) Protein Synthesis Using Weak Reductive Translation Reaction Solution(When Adding Biotin and Biotin Ligase at Time of Translation)

Transcription was conducted for the translation template DNA acquired inthe above (1) using SP6 RNA polymerase (manufactured by Toyobo Co.,Ltd.). The reaction solution contained 80 mM HEPES-KOH (pH 7.6), 16 mMmagnesium acetate, 2 mM spermidine, 10 mM DTT, NTPs (2.5 mM each), 0.8U/μl RNase inhibitor, 50 μg/ml plasmid, and 1.2 U/μl SP6 RNApolymerase/ddw 400 μl. After incubation for 2 hours at 37 ° C.,purification was conducted by phenol/chloroform extraction and passageover a Nick column (manufactured by Amersham Pharmacia Inc.), followedby ethanol precipitation, and the ethanol precipitate was dissolved in35 μl of purified water.

Translation reaction was conducted using the obtained mRNA. Thetranslation reaction solution consisted of 1.2 mM ATP, 0.25 mM GTP, 15mM creatine phosphate, 0.4 mM spermidine, 29 mM HEPES-KOH (pH 7.6), 95mM potassium acetate, 2.7 mM magnesium acetate, 0.23 mM L-form aminoacids, 0.58 U/μl RNase inhibitor (manufactured by Promega Corp.), 4nCi/μl 14C-Leu, 7.5 μg mRNA, 0.5 μM PDI, 19.5 μM biotin (Nacalai TesqueInc.), 19.5 μg/μl biotin ligase (manufactured by Avidity Inc.), and 12μl wheat embryo extract. Reaction was conducted by batch method for 3hours at 26° C. A translation reaction to which biotin was not added wasalso conducted as a control.

The reaction solution after 3 hours of translation reaction wascentrifuged for 10 minutes at 15,000 rpm to separate solubilizedcomponents, and unreacted biotin remaining therein was removed using aG-25 spin column that was equilibrated with 50 mM Tris (pH 8.0). Afterdiluting 20 μl of the spin column eluate with an equivalent amount of 50mM Tris (pH 8.0) buffer solution, 5 μl of streptavidin magnetic beads(manufactured by Promega Corp.) was added, and this was mixed gently atroom temperature. After collecting the magnetic beads using a magneticfield, supernatant fractions were acquired, and after separation bySDS-PAGE, the amount of anti-Salmonella single chain antibody wasdetermined by autoradiography. The result is shown in co-transl.biotinylation of FIG. 2. As can be seen from the figure, whentranslation was conducted in the presence of biotin and a biotin ligase(in the figure: +biotin), the amount of antibody collected by magneticbeads through bonding with streptavidin was large, and conversely, whenbiotin was not added to the reaction solution (−biotin), almost noantibodies bound to the magnetic beads. It was thus clarified thatbiotin binds to anti-Salmonella single chain antibody according to theabove-described method.

(4) Protein Synthesis Using Weak Reductive Translation Reaction Solution(When Adding Biotin and Biotin Ligase After Translation Reaction)

Biotin and a biotin ligase were added at 3 hours passage after the startof translation reaction, in a similar manner to the method ofanti-Salmonella single chain antibody described in the above (1) to (3).The results are shown in post-transl. biotinylation of FIG. 2.

As can be seen from the figure, it was found that for both the reactionsolution to which biotin was added (+) and the reaction solution towhich biotin was not added (−), almost no biotinylated antibody wasremoved with the magnetic beads. It was thus found that biotin ligaseand biotin are preferably added during the translation reaction.

Example 2 Immobilization of Biotinylated Single Chain Antibody andAnalysis of Antigen-antibody Reaction

(1) Preparation of Aldehyded Salmonella O-antigen

20 mg (2.8 μmol) of lipopolysaccharide (manufactured by Sigma ChemicalCo., Ltd.) was dissolved in 20 μL of 0.25 M sodium hydroxide aqueoussolution and stirred for 1 hour at 56° C. After dialysis againstdistilled water, 200 mg (0.8 mmol) of sodium metaperfolate was added,and this mixture was stirred for 5 minutes while shading from light.After further adding 1 ml of ethylene glycol and stirring for 1 hour,the resulting mixture was subjected to dialysis against distilled water,and the dialysis residue was lyophilized to obtain powder ofaldehyde-type Salmonella sugar chain. This powder was dissolved in 0.2ml of 20 mM sodium borate buffer (pH 9.0) (10 mg/ml). After 3 timeswashing 0.1 ml of aminated magnetic beads (NH2-Mag; manufactured byPolyscience Inc.) with 0.4 ml of the same buffer to equilibrate, thebeads were added to the above aldehyde-type Salmonella sugar chainsolution, and reaction was conducted for 6 hours at room temperature.The magnetic beads were then washed 3 times with 0.4 ml of the samebuffer. The ratio of immobilization of sugar chain onto the magneticbeads was obtained by determining the quantity of sugar chain remainedin the supernatant by a phenol/sulfuric acid process. The binding ratioof sugar chain to the magnetic beads was 40% (0.13 μmol Salmonella sugarchain/100 μl magnetic beads).

(2) Bonding of Biotinylated Anti-Salmonella Single Chain Antibody andSalmonella Sugar Chain

After synthesizing biotinylated anti-Salmonella single chain antibody bythe method described in Example 1 (total 38 μl), excess biotin wasremoved by gel filtration with a G-25 spin column that was equilibratedwith 10 mM PBST (pH 8.0) and 0.6mM CaCl₂. 40 μl of the protein solutionwas added onto a 96-well microplate together with 10 μl of immobilizedSalmonella antigen (Sal-Mag) produced in the above (1) that had beenpreviously washed with 25 μl of wheat embryo extract containing 0.6 mMCaCl₂. After gently mixing for 15 minutes, washing was performed 4 timeswith 40 μl of 0.15 M NaCl/10 mM PBST (pH 8.0), and finally elution wasconducted 4 times using an equivalent amount of 0.1 M glycine-HCL (pH2.4). Single chain antibody that had not bound to the antigen was elutedby the initial washing, and single chain antibody that had bound to theantigen was eluted by the elution conducted thereafter.

The amount of protein in each fraction (5 μl) was determined by 14 Ccount. The result is shown in FIG. 3. Here, for the purpose ofconfirming that the biotinylated single chain antibody retaine antigenspecificity, the binding result for a case of biotinylating mutant G102Din a similar manner is also shown. As can be seen from the figure, whilefor the wild type close to 50% of the total antibody was present in anactive fraction (no. 6) eluted with a pH acidic solution, in contrast,for the mutant G102D an active fraction was completely non-existent andmost of the antibody appeared in the pass-through fraction no. 1. Thisresult shows that the biotinylated anti-Salmonella single chain antibodyretained its original antigen binding activity, and thatco-translational biotinylation progresses without any loss of antigenbinding activity.

(3) Measurement of Dissociation Equilibrium Constant By BiomolecularInteraction Analysis System (Iasys)

First, streptavidin (0.1 mg/ml; manufactured by Nacalai Tesque Inc.) wasimmobilized in a biotin cuvette (manufactured by Affinity Sensors Inc.)(immobilized amount: 674 arc sec., 27.2 ng, 0.97 pMol). Next,biotinylated single chain antibody prepared in Example 1 was purifiedusing immobilized Salmonella sugar chain antigen (Sal-Mag) according tothe method described in the above (2). 8.4 μmol/50 μl of purifiedbiotinylated single chain antibody was obtained by being converted froma 14 C dpm value. This 50 μl amount was added to the above cuvette, andimmobilized on the streptavidin (immobilized amount: 433.6 arc sec.,11.5 ng, 0.4 pmol). By adding thereto various concentrations (2.4, 4.8,9.7, 12.9, 19.4 μM) of Salmonella sugar chain, association anddissociation curves were determined. FIG. 4 shows the results, whileTable 1 lists the dissociation equilibrium constant obtained from thecurves. Table 1 also lists values obtained for single chain antibodysynthesized using viable cells of Escherichia coli by the same methodfor comparison (MacKenzie, C. R. et al., J. Biol. Chem., 271, 1527-1533(1996)). As can be seen from the response curve of FIG. 4, it waspossible to immobilize biotinylated single chain antibody ontostreptavidin, and furthermore, a function for binding an antigen wasretained. As shown in Table 1, when the dissociation equilibriumconstant Kd was calculated on the basis of this curve, it was found thatthe constant was in the order of 1×10⁻⁷ to 10⁻⁸ M. Based on this resultit was clarified that the single chain antibody prepared in Example 1and immobilized by binding between biotin and streptavidin has a Kdvalue equivalent to that of complete anti-Salmonella antibody IgG. TABLE1 K_(D) K_(diss) K_(ass) antibody (M) (S⁻¹) (M⁻¹ S⁻¹) s cell-free system5.1 × 10 0.8 × 10 4.4 × 10 in-vivo system 6.5 × 10 3.1 × 10 1.8 × 10 IgG1.4 × 10 1.2 × 10 8.7 × 10

Comparative Example 1 Investigation of Immobilization EfficiencyAccording to Biotin Binding Position

Addition of Biotin to Single Chain Antibody By Chemical Bonding

The method used in this example was in accordance with antibody labelingmethods described in Immunobiochemical Methods, Biochemical ExperimentCourse, (Japanese Biochemical Society, Tokyo Kagaku Dojin (1986)).

By the method described in Example 1, a reaction solution wassynthesized without adding biotin ligase and biotin at the time oftranslation reaction, and the solution was centrifuged at 15,000 rpm for10 minutes to obtain supernatant. After diluting a 25 μl solublefraction of supernatant with an equivalent amount of 1 M sodiumbicarbonate solution, the buffer was exchanged using a G-25 Sephadexcolumn, and 1 μl of NHS-biotin (N-hydroxysuccimide-biotin, 50 mg/mlDMSO) was then added. After reacting this solution over night at 4° C.,binding ability with an antigen was analyzed as described below.Analysis of binding activity with antigen

30 μl of reaction solution prepared in the above (1) was subjected togel filtration with a G-25 spin column that was equilibrated with 10 mMof PBST (pH 8.0) and 0.6 mM of CaCl₂to remove excess biotin. 40 μl ofthe protein solution was added onto a 96-well microplate together with10 μl of immobilized Salmonella antigen (Sal-Mag) produced in Example 2(1) that had been previously washed with 25 μl of wheat embryo extractcontaining 0.6 mM of CaCl₂. After gently mixing for 15 minutes, washingwas conducted 4 times with 40 μl of 0.15 M NaCl/10 mM PBST (pH 8.0), andfinally elution was conducted 4 times using an equivalent amount of 0.1M glycine-HCL (pH 2.4). FIG. 5 shows the result. If the antibodyretained its activity it would be eluted by the latter acidic buffer,however, as can be seen from the figure, the presence of protein was notobserved in fraction numbers 10 to 13, and most of the protein waspresent in the first pass-through fraction. This result shows that thebiotinylated single chain antibody produced by the aforementionedchemical process lost its antigen-binding activity.

Example 3 Production of Single Chain Antibody Inserted WithPolyhistidine Peptide and Immobilization Thereof

(1) Production of Single Chain Antibody Containing Polyhistidine Peptidein a Linker Part

PCR was conducted employing scfv-pEU described in Example 1 (1) as atemplate, and using LA Taq (manufactured by Takara Co., Ltd.) kit withprimers comprising the nucleotide sequences represented by SEQ ID NOS: 8and 9. The PCR reaction solution was prepared with 5 μl of 10× LAbuffer, 5 μl of 25 mM magnesium chloride, 8 μl of 2.5 mM dNTP, 1 μl of20 μM primer (for each primer), and 0.1 ng of template plasmid/50 μl.The reaction was conducted by heating at 94° C. for 1 min×1 cycle, 94°C. for 45 sec/55° C. for 1 min/72° C. for 1.5 min×30 cycles, and then72° C. for 5 min. In accordance with a conventional method, the ends ofamplified DNA fragments were blunted using KOD T4 polymerase(manufactured by NEB Inc.), the fragments were phosphorylated withPolynucleotide Kinase (NEB Co., Ltd.), and self-ligation was thencarried out using Ligation High (manufactured by Toyobo Co., Ltd.) toproduce a circular plasmid (FIG. 1; hereafter, this is sometimesreferred to as “scFv-pHIS-pEU”).

After conducting transcription according to the method described inExample 1 (3) employing this plasmid as a template and purifying themRNA, DTT in the translation reaction solution was replaced with 200 μMof mercaptoethanol to conduct a translation reaction. The reactionsolution after 3 hours of translation reaction was centrifuged for 10minutes at 15,000 rpm to separate solubilized components, and excessmercaptoethanol was removed using a G-25 spin column that wasequilibrated with 50 mM phosphate buffer (pH 7.0), 500 mM NaCl, and 5%glycerol (binding buffer).

After diluting 20 μl of the spin column eluate with an equivalent amountof the binding buffer, 80 μl of the solution was passed over a 200 μlnickel column (50% resin) (metal affinity resin, Talon) that waspreviously washed 6 times with 150 μl of binding buffer, and this wasincubated for one hour at room temperature. This column was washed 4times (w1 to w4 in the figure) with 150 μl of 50 mM phosphate buffer (pH7.0), 500 mM NaCl, 5% glycerol, and 6 mM imidazole (washing buffer), andelution was then carried out 5 times (e1 to e5 in the figure) with 150μl of 50 mM phosphate buffer (pH 7.0), 500 mM NaCl, and 150 mM imidazole(elution buffer) The amount of single chain antibody contained in eachfraction was measured by 14 C dpm value. FIG. 6 shows the result. In thefigure, C indicates the 14 C dpm value in the total amount ofprotein-containing solution prior to passage over the column. Thehorizontal axis of the graph shows fraction numbers, w1 to w4 show the14 C dpm values in fractions eluted by the washing buffer, while e1 toe5 show the 14 C dpm values in fractions eluted by the elution buffer.ET shows the total of the 14 C dpm values for e1 to e5.

Fraction numbers e1 to e5 indicate the existence of single chainantibody containing polyhistidine peptide binding specifically withnickel. As can be seen from the figure, it was found that approximately50% of the total synthesized amount of single chain antibody could bepurified by nickel column. It was confirmed by the method described inExample 2 that the purified single chain antibody also retainedantigen-binding activity. This result indicates that a single chainantibody having polyhistidine peptide incorporated in a linker partthereof can be immobilized to a nickel solid phase in a condition inwhich it retains its activity.

INDUSTRIAL APPLICABILITY

According to the present invention, there is provided a single chainantibody or labeled single chain antibody that retains activity forbinding specifically with an antigen. The single chain antibody can bebound to a solid phase via a labeling substance, and can be used toproduce an antibody chip or the like. By synthesizing this single chainantibody using a cell-free protein translation system that allows anintramolecular disulfide bond to be retained, there can be provided anantibody having specific binding ability against an antigen that ishigher than that of an antibody synthesized within a viable cell such asEscherichia coli.

1. A labeled single chain antibody, wherein the antibody carries alabeling substance in a linker part of a single chain antibody.
 2. Thelabeled single chain antibody of claim 1, carrying a labeling substancein a linker part of a single chain antibody, wherein a heavy chain and alight chain of the antibody are variable regions.
 3. The labeled singlechain antibody of claim 1, having a structure in which a heavy chain anda light chain of an antibody are crosslinked through a linker, andcarrying a labeling substance in the linker part, wherein the labelingsubstance is a substance that is capable of binding to a polypeptide ofthe linker part of the antibody in the presence of a specific enzyme. 4.The labeled single chain antibody of claim 1, having a structure inwhich a heavy chain and a light chain that are variable regions of theantibody are crosslinked through a linker, and carrying a labelingsubstance in the linker part, wherein the labeling substance is asubstance that is capable of binding to a polypeptide of the linker partof the antibody in the presence of a specific enzyme.
 5. The labeledsingle chain antibody of claim 1, having a structure in which a heavychain and a light chain of an antibody are crosslinked through a linker,and carrying a labeling substance in the linker part, wherein thelabeling substance is incorporated as one part of the linker part of theantibody.
 6. The labeled single chain antibody of claim 1, having astructure in which a heavy chain and a light chain that are variableregions of the antibody are crosslinked through a linker, and carrying alabeling substance in the linker part, wherein the labeling substance isincorporated as one part of the linker part of the antibody.
 7. Thelabeled single chain antibody of claim 1, having a structure in which aheavy chain and a light chain of the antibody are crosslinked through alinker, and carrying in the linker part a labeling substance that iscapable of binding to a polypeptide of the linker part of the antibodyin the presence of a specific enzyme, wherein the labeling substance isbiotin and the enzyme is a biotin ligase.
 8. The labeled single chainantibody of claim 1, having a structure in which a heavy chain and alight chain that are variable regions of the antibody are crosslinkedthrough a linker, and carrying in the linker part a labeling substancethat is capable of binding to a polypeptide of the linker part of theantibody in the presence of a specific enzyme, wherein the labelingsubstance is biotin and the enzyme is a biotin ligase.
 9. The labeledsingle chain antibody according to of claim 1, which has a Kd value thatis equivalent to a Kd value of a naturally occurring antibody and whichis produced by a cell-free protein translation system using wheatembryo. 10-11. (canceled)
 12. A DNA in which DNAs encoding a heavy chainand a light chain of an antibody having binding ability against aspecific antigen are linked through a DNA encoding a linker, wherein theDNA encoding a linker comprises a nucleotide sequence that is capable ofbinding with a labeling substance in the presence of a specific enzymeafter translation.
 13. The DNA of claim 12, in which DNAs encoding aheavy chain and a light chain that are variable regions of an antibodyhaving binding ability against a specific antigen are linked through aDNA encoding a linker, wherein the DNA encoding a linker comprises anucleotide sequence that is capable of binding with a labeling substancein the presence of a specific enzyme after translation.
 14. The DNA ofclaim 12, in which DNAs encoding a heavy chain and a light chain of anantibody having binding ability against a specific antigen are linkedthrough a DNA encoding a linker that comprises a nucleotide sequencethat is capable of binding with a labeling substance in the presence ofa specific enzyme after translation, wherein the nucleotide sequencethat is capable of binding with a labeling substance encodes an aminoacid sequence that is recognized by a biotin ligase.
 15. The DNA ofclaim 12, in which DNAs encoding a heavy chain and a light chain thatare variable regions of an antibody having binding ability against aspecific antigen are linked through a DNA encoding a linker thatcomprises a nucleotide sequence that is capable of binding with alabeling substance in the presence of a specific enzyme aftertranslation, wherein the nucleotide sequence that is capable of bindingwith a labeling substance encodes an amino acid sequence which isrecognized by a biotin ligase.
 16. A method for producing a labeledsingle chain antibody, wherein the DNA according to claim 12 issubjected to transcription and translation utilizing a protein synthesissystem in the presence of a labeling substance and a specific enzyme.17. (canceled)
 18. The method for producing a labeled single chainantibody according to claim 16, wherein the protein-synthesis system isa wheat embryo-derived cell-free protein translation system, and aconcentration of a reducing agent in a translation reaction solutionthereof is a concentration whereby a disulfide bond of a labeled singlechain antibody to be produced is retained and cell-free proteinsynthesis is enabled.
 19. The method for producing a labeled singlechain antibody according to claim 18, wherein the method is conducted inthe presence of an enzyme that catalyzes a disulfide bond exchangereaction.
 20. A labeled single chain antibody which has a Kd value thatis equivalent to a Kd value of a naturally occurring antibody and isproduced by the method for producing a labeled single chain antibodyaccording to claim 19, utilizing a wheat embryo-derived cell-freeprotein translation system.
 21. A method for producing an immobilizedsingle chain antibody, wherein any one of the antibodies describedhereunder is brought into contact with a reaction platecompartmentalized into a plurality of regions having on the surfacethereof a substance that binds specifically with a labeling substance ofthe antibody: 1) a labeled single chain antibody of claim 1, wherein theantibody has a structure in which a heavy chain and a light chain of theantibody are crosslinked through a linker and the antibody carries alabeling substance in the linker part; 2) a labeled single chainantibody having a structure in which a heavy chain and a light chain ofthe antibody are crosslinked through a linker, and carrying a labelingsubstance in the linker part, wherein the heavy chain and the lightchain of the antibody are variable regions; 3) a labeled single chainantibody having a structure in which a heavy chain and a light chain ofthe antibody are crosslinked through a linker, and carrying a labelingsubstance in the linker part, wherein the labeling substance is asubstance that is capable of binding to a polypeptide of the linker partof the antibody in the presence of a specific enzyme; 4) a labeledsingle chain antibody having a structure in which a heavy chain and alight chain that are variable regions of the antibody are crosslinkedthrough a linker, and carrying a labeling substance in the linker part,wherein the labeling substance is a substance that is capable of bindingto a polypeptide of the linker part of the antibody in the presence of aspecific enzyme; 5) a labeled single chain antibody having a structurein which a heavy chain and a light chain of the antibody are crosslinkedthrough a linker, and carrying a labeling substance in the linker part,wherein the labeling substance is incorporated as one part of the linkerpart of the antibody; 6) a labeled single chain antibody having astructure in which a heavy chain and a light chain that are variableregions of the antibody are crosslinked through a linker, and carrying alabeling substance in the linker part, wherein the labeling substance isincorporated as one part of the linker part of the antibody; 7) alabeled single chain antibody having a structure in which a heavy chainand a light chain of the antibody are crosslinked through a linker, andcarrying in the linker part a labeling substance that is capable ofbinding to a polypeptide of the linker part of the antibody in thepresence of a specific enzyme, wherein the labeling substance is biotinand the enzyme is a biotin ligase; 8) a labeled single chain antibodyhaving a structure in which a heavy chain and a light chain that arevariable regions of the antibody are crosslinked through a linker, andcarrying in the linker part a labeling substance that is capable ofbinding to a polypeptide of the linker part of the antibody in thepresence of a specific enzyme, wherein the labeling substance is biotinand the enzyme is a biotin ligase.
 22. The method for producing animmobilized single chain antibody of claim 21, wherein two or more kindsof different immobilized single chain antibodies are immobilized on areaction plate compartmentalized into a plurality of regions.
 23. Theproduction method according to claim 21, wherein a labeling substance isbiotin and a substance that binds specifically with the labelingsubstance is streptavidin.
 24. An immobilized single chain antibodyprepared by the production method according to of claim
 21. 25. A methodfor analyzing an antigen-antibody reaction, wherein a test substance isbrought into contact with the immobilized single chain antibody of claim24, and binding ability of the test substance against the immobilizedsingle chain antibody is analyzed.
 26. A method for analyzing anantigen-antibody reaction, comprising the steps of: (1) preparing alabeled single chain antibody under conditions in which a disulfide bondof a single chain antibody is retained, comprising the step of thefollowing (i) or (ii): (i) producing a labeled single chain antibody bysubjecting a DNA, in which DNAs encoding a heavy chain and a light chainof an antibody having binding ability with a specific antigen are linkedthrough a DNA encoding a linker comprising a nucleotide sequence that iscapable of binding with a labeling substance in the presence of aspecific enzyme after translation, to transcription and translationutilizing a wheat cell-free protein synthesis system in the presence ofa specific enzyme; or (ii) producing a labeled single chain antibody bysubjecting a DNA, in which DNAs encoding a heavy chain and a light chainthat are variable regions of an antibody having binding ability with aspecific antigen are linked through a DNA encoding a linker comprising anucleotide sequence that is capable of binding with a labeling substancein the presence of a specific enzyme after translation, to transcriptionand translation utilizing a wheat cell-free protein synthesis system inthe presence of a specific enzyme; (2) preparing a substance (adaptersubstance) that binds specifically with a labeling substance of alabeled single chain antibody in a case where the labeling substance ofthe labeled single chain antibody is an immobilizing substance,comprising the steps of: (i) immobilizing a substance (adaptersubstance) that binds specifically with a labeling substance of alabeled single chain antibody to a reaction plate compartmentalized intoa plurality of regions; (ii) removing a substance (adapter substance)that binds specifically with a labeling substance of a labeled singlechain antibody that was not immobilized to the reaction plate in thepreceding (i); and (iii) before and after the step of the preceding (i)or (ii), removing nonspecific adsorption from the reaction plate asappropriate; (3) preparing an immobilized labeled single chain antibodyin a case where a labeling substance of the labeled single chainantibody is an immobilizing substance, comprising the steps of: (i)adding a required amount of the labeling substance of the labeled singlechain antibody prepared in (i) or (ii) of the above (1) onto a reactionplate compartmentalized into a plurality of regions having a substance(adapter substance) of (2) that binds specifically with the labelingsubstance of the labeled single chain antibody on the surface thereof,whereby to contact; (ii) removing a labeled single chain antibody thatwas not immobilized to the substance (adapter substance) that bindsspecifically to the labeled single chain antibody on the reaction platein the preceding (i); and (iii) following the preceding step (ii),removing nonspecific adsorption from the reaction plate as appropriate;(4) preparing a labeled single chain antibody in a case where a labelingsubstance is a signal substance, comprising the steps of: (i) removingnonspecific adsorption from a reaction plate compartmentalized into aplurality of regions as appropriate; and (ii) adding a required amountof the labeling substance of the labeled single chain antibody preparedin (i) or (ii) of the above (1) onto the reaction plate; (5) adding arequired amount of a test substance onto each reaction plate accordingto the above (3) or (4), and analyzing the binding ability of a labeledsingle chain antibody with the test substance; and (6) based on thebinding ability result obtained in the above (5), qualitatively orquantitatively determining the interaction between the labeled singlechain antibody and the test substance.
 27. A reagent kit for measuringan antigen-antibody reaction, comprising a reagent to be used in theanalysis method according to claim
 25. 28. An immobilized single chainantibody that has a Kd value that is equivalent to a Kd value of anaturally occurring antibody and that is produced by the method forproducing an immobilized single chain antibody according to claim 21utilizing a wheat embryo-derived cell-free protein translation system.